The Role Of Nucleolin Expression In Apoptosis Of Human Osteosarcoma Cells

Chapter one:Expression of nucleolin and bcl-2in human osteosarcoma cellsObjective1.wild-type p53human osteosarcoma cell line U-2OS,p53-null human osteosarcoma cell line Saos-2,p53-mutant human osteosarcoma cell line MG-63and the immortalized cell line hFOB1.19are choosen in order to understand expressions of nucleolin and bcl-2. For the purpose of understand its role in apoptosis,it is the basement that nucleolin and bcl-2expressions of these cells are detected in qualitation.2.U-2OS,Saos-2and MG-63are choosen in order to understand inhibition of proliferation induced by cisplatin in human osteosarcoma cells. For the purpose of suitable density of Cisplatin is choosen.Methods1.At exponential growth phases,RNA of U-2OS,Saos-2,MG-63and hFOB1.19are extracted and reversed into cDNA. PCR primers aimed at nucleolin,bcl-2and β-actin are designed respectively. The best conditions of PCR detection are determined.The Products of PCR are analysed by gel electrophoresis. 2.At exponential growth phases, protein of U-2OS> Saos-2^MG-63and hFOB1.19are extracted respectively. Expression of nucleoln,bcl-2and β-actin is detected by Western bloting.3.U-2OS,Saos-2and MG-63cells were treated with different concentrations of cisplatin, then cell viability was analyzed with MTT assays; Growth curve was drawn to get a suitable concentration as experimental concentration.Results1. Compared withnormal osteoblastic cell,overexpressions of nucleolin and bcl-2can be detected in osteosarcoma cells U-2OS,Saos-2and MG-632. The influence of cells inhibition in proliferation induced by cisplatin have a lot to do with the Expression of nucleolin and bcl-2in human osteosarcoma cellsConclusion1. Overexpressions of nucleolin and bcl-2are detected in U-2OS(wild-type p53),Saos-2(p53-null) and MG-63cells(p53-mutant).The results show overexpression of nucleolin and bcl-2in these three kind of cell lines regardless of P53expression.2.In our experiment, Western bloting and RTPCR conditions are suitable and feasible;3.Features of nucleolin and bcl-2expressions are accord with other studies, proving feasiblility of this experiment,providing supports for further studies, which are researches about therapeutic effects of nucleolin antisense oligonucleotides and its correlation with stabilization of bcl2mRNA.4. Effect of cisplatin on osteosarcoma cells viability are concerned with overexpressions of nucleolin and bcl-2. Chaper two:Recombinant antisense nucleolin oligonucleotides sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent mannerObjectiveTo investigate the effect of nucleolin down-regulation on inhibition of proliferation and apoptosis induced by cisplatin in human osteosarcoma cells.Methods1.Targeted RODN and ASODN of nucleolin were designed and synthesized,and then transfected to osteosarcoma cells U-2OS^Saos-2and MG-63;then efficacy evaluation of RODN and ASODN were detected by Immunohistochemical and Immunoblotting;2.each human osteosarcoma cells were divided to six groups randomly:control group; CDP group; RODN group; RODN+CDP group; ASODN group and ASODN+CDP group; Cell growth suppression of each group was analyzed by MTT assay; RT-PCR and Immunoblotting were used to detected the expression of nucleolin and Bcl-2mRNA and protein in each group; Morphologic character of apoptosis was evaluated by Hoechst33258staining; The percentage of apoptotic cell was analyzed by flowcytometer assay; caspase-3activity were used to detected the apoptosis of each group.Results1.ASODN can inhibit remarkably the expression of nucleolin mRNA and protein in U-2OS,Saos-2and MG-63cells;2.ASODN can inhibit remarkably the expression of bcl-2mRNA and protein in U-2OS,Saos-2and MG-63cells;2. The proliferation of U-2OS,Saos-2and MG-63cells were inhibited remarkably in ASODN+CDP group compared with CDP group3. The expression of nucleolin and Bcl-2mRNA and protein were weaker in ASODN+CDP group compared with CDP group4.Nucleolin antisense oligonucleotides could promote cisplatin-mediated apoptosis in U-2OS,Saos-2and MG-63cells with a higher percentgae of apoptotic cells than CDP group regardless of P53expression.ConclusionRecombinant antisense nucleolin oligonucleotides sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner Chaper three:Overexpression of nucleolin in human osteos arco ma cells induces stabilization of bcl2mRNAObjectiveTo investigate the overexpression of nucleolin in human osteosarcoma cells and its correlation with stabilization of bcl2mRNA.Methods1. After treated with Actinomycin D,the stabilization of bcl2mRNA was evaluated by RT-PCR in U-2OS,Saos-2,MG-63and hFOB1.19cells;2. The role of nucleolin bind to bcl2mRNA in U-2OS,Saos-2, MG-63and hFOB1.19cells were evaluated by RNA-CHIP;3.Recombinant antisense nucleolin oligonucleotides is transfected into human osteosarcoma cells MG-63,and the effects of it on the stabilization of bcl2mRNA and the role of nucleolin bind to bcl2mRNA was evaluated.Results1.Overexpression of nucleolin and Bcl2proteins in human osteosarcoma cells compared with human osteoblast cells;2.Bcl2mRNA stability is increased in human osteosarcoma cells relative to human osteoblast cells;3.Binding of Bcl2mRNA to nucleolin is greater in extracts of human osteosarcoma cells compared with extracts of human osteoblast cells;4.Knockdown of nucleolin decreases Bcl2mRNA stability and Binding of Bcl2mRNA to nucleolin in human osteosarcoma MG63cells.ConclusionOverexpression of nucleolin in human osteosarcoma cells induces stabilization of bcl2mRNA...